Journal of Electron Microscopy Advance Access originally published online on January 13, 2006
Journal of Electron Microscopy 2005 54(6):519-528; doi:10.1093/jmicro/dfi070
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Morphological and functional analysis of Rac1B in Dictyostelium discoideum
1 Indiana University South Bend 1700 Mishawaka Avenue, South Bend, IN 46634, USA
2 University of Southern Indiana Evansville, IN 47712, USA
*To whom correspondence should be addressed. E-mail: ropope{at}iusb.edu
Rac1B, a small GTP-binding protein in Dictyostelium discoideum, is involved in regulation of the actin cytoskeleton. Scanning electron microscopy revealed distinctive phenotypes for the wild-type, constitutively active, constitutively inactive and overexpressing cell lines. Immunofluorescence showed constitutively active Rac1B localized to lamellipodia and sites of cell-to-cell contact. In contrast, constitutively inactive Rac1B was homogenously distributed throughout the cell. Phalloidin staining demonstrated that active Rac1B co-localizes with F-actin. Amoebae expressing mutant Rac1B exhibited defects in endocytosis, cytokinesis and multicellular development. Overexpression of wild-type Rac1B positively affected fluid-phase endocytosis, whereas expression of either constitutively active or inactive forms of Rac1B inhibited endocytic rates. The greatest defects in cytokinesis were observed in amoebae producing constitutively active Rac1B or overexpressing wild-type Rac1B. These cells were severely multinucleated and divided by traction-mediated cytofission when placed onto a solid surface. Cells expressing mutant Rac1B were unable to form viable fruiting bodies. Elucidating the role of Rac1B in filamentous actin dynamics will lead to a better understanding of cell adhesion, development and cell motility.
Keywords Rac1B, Rac, Rho, Dictyostelium, actin, cytoskeleton
Received 3 October 2005, accepted 20 December 2005